Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based...
-
Upload
maurice-mcgee -
Category
Documents
-
view
214 -
download
0
Transcript of Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based...
![Page 1: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/1.jpg)
Functionality of microbial phenotypic heterogeneity in bioprocessing
conditions: an analysis based on the use of on/off-line flow cytometryDelepierre Anissa1, Brognaux Alison1, Baert Jonathan1, Delvigne
Frank1.1 University of Liège, Gembloux Agro Bio Tech
Microbial processes and interactions - Belgium
![Page 2: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/2.jpg)
Negative impact of microbial phenotypic heterogeneity on process productivity but improvement of robustness?
Accepted picture : only a fraction of the population (non productive phenotypes) affects the global production profile
Time
Met
abo
lite
co
nce
nta
tio
n
Global production profile
Non productive phenotypes
Productive phenotypes
Microbial phenotypic heterogeneity ?
![Page 3: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/3.jpg)
Phenotypic heterogeneity
o Noise (Intrinsic and extrinsic noise)o Reinforced by external noise: spatial heterogeneity
Patnaik PR, (2006) External, extrinsec and intrinsec noise in cellular systems : analogies and implications for protein synthesis. Biotechnology and Molecular Biology Review Vol. 1 (4), pp, 121-127.
![Page 4: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/4.jpg)
How to track this phenotypic heterogeneity?
By combining flow cytometry, genetically encoded biosensors and external dyes
Genetically encoded: GFP
Exogenous: Propidium iodide
Flow cytometry and cell sorting session
Brognaux Alison
![Page 5: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/5.jpg)
The flow cytometry
![Page 6: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/6.jpg)
QFC
FCM
Manual sampling
QFC
FCM
Sampling pump
On-line
Flow cytometry (FCM) : different modes of operation
Real timeBrognaux, A., Han, S., Sorensen, S. J., Lebeau, F., Thonart, P., Delvigne, F., (2013) A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors. Microbial cell factories 12, 1475-2859
![Page 7: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/7.jpg)
Application of flow cytometry
Evaluation of E. coli outer membrane permeability
![Page 8: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/8.jpg)
Bioprocess conditions: Limitation of carbon substrate
Glucose uptake: 2 barriers Modulation of membrane permeability: Self-preservation and nutritional competence SPANC
Phenotypic heterogeneity
Ferenci T. (2005) Maintaining a healthy SPANC balance through regulatory and mutational adaptation. Mol. Microbiol. 2005;57:1-8
Shimizu K(2013) Regulation systems of bacteria such as escherichia coli in response to nutrient limitation and environmental stresses. Metabolites 2013, 4(1):1-35.
![Page 9: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/9.jpg)
Bet hedging
Exogenous dye: Propidium iodide staining Hypothesis : accumulation of PI in the periplasm when cells are exposed to substrate limitation (increased expression of porins)
How to track the Porin-mediated outer membrane permeability?
OmpF OmpC
LamB Extracellular env.
Periplasm
Cytoplasm
Cut-off < 800 DaCut-off < 600 DaPI (MW = 668
Da)
PI
![Page 10: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/10.jpg)
Intermediate PI uptake : Propidium iodide (PI) uptake and appearance of an intermediate subpopulation with reduced red fluorescence
Fed-batch, well-mixed reactor
![Page 11: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/11.jpg)
Outer membrane permeabiliy in metabolic engineered E. coli k-12 strains?
Impact on robustness and phenotypic heterogeneity?
ΔLamBΔOmpFΔOmpC
single-gene Knockout deletion (KEIO collection)
![Page 12: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/12.jpg)
Test in microwell plate: Batch culture
Comparaison of microbial growth kinetic
Spectramax
On-line measuring of OD600nm
![Page 13: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/13.jpg)
Sampling at stationary phase: PI staining and FC analysis
10g/L
1g/L
![Page 14: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/14.jpg)
Test in mini-bioreactor: batch culture
8-15 ml
On-line measure of pH and Dissolved oxygen by optical sensors
8 mini-bioreactors in parallel
![Page 15: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/15.jpg)
Sampling at exponential and stationary phase: OD600nm
0h 6h 13h 24h 29h 47h0
2
4
6
8
10
12
14
Biomass production
WT ΔLamB ΔOmpF ΔOmpC
Time of culture
OD
600nm
![Page 16: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/16.jpg)
6h 13h 24h 29h 47h
WT
Sampling at exponential and stationary phase: PI staining and FC analysis
Exponential phase
Stationary phase
Membrane permeability increasing
![Page 17: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/17.jpg)
Comparaison of strains in stationary phase (24h)
WT ΔLamBΔOmpFΔOmpC
![Page 18: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/18.jpg)
ΔOmpC : increased level of intermediate PI+ sub-population
High cut-off porins upregulation?
High outer membrane permeability
Efficient nutrition Low resistance
Lower biomass production
CONCLUSIONS
![Page 19: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/19.jpg)
ΔLamB: Decreased level of intermediate PI+ sub-population
LamB: Major implication in the outer membrane permeability related to substrate limitation?
Low outer membrane permeability
Poor nutrition High resistance
Lower biomass production
![Page 20: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/20.jpg)
Thank you for your attention
![Page 21: Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on/off-line flow cytometry Delepierre.](https://reader034.fdocuments.us/reader034/viewer/2022042717/56649d945503460f94a7c11d/html5/thumbnails/21.jpg)
What’s next?
• Omics approach on the subpopulation by cell sorting: Stress response?